Method to detect bacteria

ABSTRACT

The present invention relates to a method for enhancing the time of response of an assay for a first bacterium, wherein: a) the first bacterium is exposed to infection by phage particles to which the first bacterium is permissive; b) the infected bacterium is treated to inactivate exogenous phage particles; c) the treated bacterium is cultivated in the presence of a second bacterium which is permissive to infection by the phage or its replicand and which has a doubling rate greater than the effective doubling rate of the first bacterium; and d) assessing the extent of plaque formation and/or of second bacterium growth in the cultivated second bacterium cells. The method can be used to assess the presence of first bacterium in a sample, notably where the first bacterium is a slow growing bacterium, such as Mycobacterium tuberculosis, where the method enables an operator to detect the presence of low amounts of the bacterium in sample within days instead of weeks as required by conventional cultivation techniques. The invention can also be used to assess the effect of a drug or other treatment on a bacterium or on a virus. The invention also provides a diagnostic kit for use in the method of the invention.

This application is the US national stage entry under 35 U.S.C. 371 ofinternational application PCT/GB96/03097, filed Dec. 16, 1996.

The present invention relates to a method, notably to a method fordiagnosing the presence of a bacterium in a sample.

BACKGROUND TO THE INVENTION

It is common practice to propagate bacteria from a sample of a bodilyfluid, such as sputum, on a suitable culture medium so as to be able todetect the colonies of bacteria on the culture medium either visually orusing a test reagent or assay. Such a technique is applicable over awide range of bacteria and has become widely accepted as a standardbacteria detection and diagnostic procedure. However, problems arisewhere the bacterium is a slow growing type where it may take severaldays or even weeks before adequate growth of the bacteria has takenplace to be detectable. This is particularly the case with someMycobacterium or Legionella species, where it can take up to four weeksor more to propagate detectable colonies of the bacteria. This enforcesan unacceptable delay in confirming infection with the bacteria inpatients and it would be desirable to provide a more rapid assessment ofthe presence and identity of the bacteria.

It has been proposed in PCT Application No WO92/02633 to prepare asample of a viable bacterium and to infect that with a strain of a viruswhich is specific to that bacterium, such bacteria-infecting virus beingdenoted as bacteriophage or, more commonly, phage. Some of the phageparticles infect the bacteria and replicate within the bacterial cell.Those phage particles which are not absorbed by the bacteria remain withthe mother liquor or matrix of the carrier of the bacteria, typically aculture medium. If the infected sample is then treated, for example withheat and/or an acid to kill, or is washed with a surfactant to remove,the exposed, exogenous, phase particles, the exogenous phage particlesare inactivated or removed. The infecting, endogenous, phage particlesare not inactivated, since they are protected within the bacterial cell.The treated infected bacteria can then be cultured in the presence offurther un-infected bacteria which are permissive to, that is can beinfected by, the phage particles. The phage particles within theinfected bacteria replicate causing the cell wall to rupture or lyse andrelease its load of replicated phage particles. This first generation ofprogeny phage particles infects other bacterial cells, causingsuccessive cycles of infection replication and lysis. This gives anexponential increase in the number of phage particles in the culturemedium. Either the reduced growth of the bacterial cells or the largenumber of replicated phage particles can be measured or observed, evenwhere the initial number of infected bacterial cells was small, thusenhancing the sensitivity of the detection of the presence of theinitial bacterial cells in the sample being assessed.

This modification enhances the sensitivity of detection of bacterialcells and has been used successfully in the detection of fast growingbacteria such as Salmonella spp. However, with slow growing bacteriasuch as Mycobacteria spp, we have found that the time taken for thisprocedure to give a detectable population of phage particles remainssubstantially the same as with other techniques and the procedure doesnot solve the time problem with such bacteria. The need still exists fora procedure which achieves rapid and accurate detection of slow growingbacteria.

We have devised a method by which slow growing bacteria can be rapidlydetected, thus overcoming the problems in having to wait days or evenweeks for such detection. The method can also be used to determine thesensitivity of given bacteria to antibiotic and other drugs and todetermine whether a given antibiotic or drug is active against givenbacteria or whether a given virucidal composition is effective against agiven virus. Since the method can be performed using simple apparatusand by unskilled persons, the method readily lends itself to operationin third world countries where skilled microbiologists and complexapparatus may not be available.

SUMMARY OF THE INVENTION

Accordingly, from one aspect the present invention provides a method fordetecting the presence of first bacterium in a sample, which methodcomprises the steps of:

a. infecting a viable sample of the first bacterium cells with a phagewhich is specific to that bacterium whereby at least some of thebacterium cells each absorb one or more of the phage particles;

b. in-activating or removing the phage particles which have not beenabsorbed by the bacterium cells;

c. cultivating the infected first bacterium cells in a medium containingat least one other second bacterium which has a reproduction rategreater than the reproduction rate of the said first bacterium wherebyat least some of the said second bacterium cells become infected byphage particles released upon the lysis of said infected first bacteriumcells, each of said infected bacterium cells acting as a source forfurther infection of a subsequent generation of the infection cycle; and

d. monitoring either the population of replicated phage particles and/orthe reduced growth of the said second bacterium in step c to determinethe presence of said first bacterium cells in the sample under test.

Preferably, the cultivation and infection of the second bacterium iscarried out to or near the steady state at which further bacterial cellgrowth and lysis substantially ceases. At this state, a substantiallysteady population of dead bacteria cells due to the activity of thephage particles is present in the cultivation medium. The areas of thecultivation medium in which phage replication has taken place can bedetected visually as clear spots in the surrounding cultivation mediumwhich is more cloudy, opaque or otherwise visually different due to thegrowth of viable bacteria. These areas of reduced bacterial growth dueto phage activity are commonly termed plaques. Since the secondbacterium reproduces at a greater rate than the first bacterium, thetime taken for the initial infected first bacterium cells to producedetectable plaques is greatly reduced. This acceleration in thedevelopment of plaques does not occur when the same bacterium is usedfor the initial phage infection and for the subsequent cultivationsteps.

Alternatively, the presence of replicated phage particles in thecultivation medium at the end of step c can be assessed usingconventional phage assay techniques. This can be done in addition to oras an alternative to the determination of the existence of plaques asdescribed above.

The method of the invention can thus be applied to the detection of slowgrowing bacteria in a sample, for example from a patient suspected ofbeing infected with Mycobacterium tuberculosis or Legionnaires disease,where a conventional assessment would takes days or weeks to give anyobservable indication that such bacteria were present in the initialsample. In such a case, a proportion of the initial sample is infectedwith a phage specific to the expected bacterium and the phage particlesare carried through the in-activation step b because they are protectedby the bacterial cells which they have infected. These protected phageparticles cause the development of phage or plaques in the environs ofeach infected first bacterium cell in the cultivation of the infectedcells--step c. If none of the suspected bacteria were present in thesample, substantially no phage or plaques would be formed in step c andthe cultivation medium would present a substantially uniform appearance.

The invention may also be carried out using a known bacterium which hasbeen treated with an antibiotic or other drug under test to determinewhether the antibiotic or drug has any bactericidal effect. In thiscase, the population of phage or plaques at the end of step c indicatesthe efficacy or otherwise of the antibiotic or drug in killing theinitial bacterium. In a conventional test method, the antibiotic or drugwould be applied to the bacterium and the number of dead cells would bea direct indication of the efficacy of the antibiotic or drug. However,it may be difficult to observe the individual dead cells except under amicroscope. In this application of the invention, the treatment of thefirst bacterium with the antibiotic or drug, which may kill the firstbacterium, is combined with infection of the remaining viable firstbacterium with the phage particles to demonstrate how many of the firstbacteria have escaped being killed by the antibiotic or drug.

In another application of the invention, a sample of a virus, or phage,is treated with a composition which has a virucidal or virus growthregulant effect. The treated phage is then used to infect a viable firstbacterium which is permissive to that specific phage, that is thebacterium can be infected by that phage. The infected bacterium is thentreated to in-activate the exogenous phage particles, that is thosewhich do not infect the bacterium cells and remain exposed outside, orexogenous to, the cells. The in-activated infected first bacterium cellmaterial is then cultured in the presence of a second, fasterreproducing, bacterium to produce plaques where the phage particles inthe infected first cells have replicated and lysed. If the initialtreatment of the phage particles has been fully effective, no viablephage particles will be present when the first bacterium material isinfected and, therefore, no viable phage particles will carry through tothe cultivation in the presence of the second bacterium. Therefore, thesecond bacterium will reproduce to give a substantially uniform colouror opacity to the culture medium. If, however, the initial treatment ofthe phage particles was not fully effective, phage particles will becarried through to the cultivation of the second bacterium and willcause plaques, which can be detected visually.

For convenience, the invention will be described hereinafter in terms ofthe assessment of the presence of a slow growing Mycobacterium spp. Theinvention can be applied to bacteria from a wide range of aminal orvegetable hosts, but is of especial application in the detection ofbacteria from mammal hosts and for convenience the invention will bedescribed with especial reference to the assessment of such a slowgrowing bacterium in an initial sample of a bodily material from a humansuspected of being infected with that bacterium.

The term slow growing bacterium is used herein to denote those bacteriawhich, under normal conditions of propagation at 37° C. in a culturemedium comprising 90% v/v of the medium known as 7H9 and 10% v/v of thenutrient known as OADC, have an effective reproduction cycle as assessedby a doubling of the bacterial cell population (the effective doublingrate) in excess of 10 hours. Such bacteria would normally take in excessof 72 hours using a conventional cultivation technique to producedetectable phage population or plaques, or are diffiult to cultivate.Using the method of the invention, phage or plaques can usually bedetected within 16 hours with such bacteria.

Typical slow growing bacteria include Mycobacterium spp, notablyMycobacterium tuberculosis, Mycobacterium paratuberculosis andMycobacterium avium; Legionella spp, notably Legionella pneumophila;Helicobacter spp, notably Helicobacter pylori; Streptococcus spp,notably Streptococcus adjacens; and Rikettsia spp, notably Rickettsiaprowazekii. The invention may also be applied to bacteria which, whilsthaving an instantaneous doubling rate of less than 10 hours, have anaverage effective doubling rate greater than this due to the difficultywith which they initially propagate, for example Streptococcus defectensand Extremophiles spp. The term slow growing bacteria is therefore usedherein and in the claims to denote bacteria which are inherently slowpropagators and bacteria which have a slow average propagation rate overa period of ten hours due to intermittent failure or interruptions intheir propagation cycle.

Whilst the invention is of especial benefit in detecting such slowgrowing bacteria, the invention can be applied to any bacterium where itis desired to increase the speed of formation of a detectable phagepopulation or plaques, for example in the detection of bacteria whichhave a doubling rate shorter than 10 hours, for example in the range 4to 10 hours, in human samples, food products or in samples taken todetermine bacterial infestations in kitchens and the like, so as toprovide more rapid testing for bacteria than would be achieved usingconventional bacterial cultivation techniques. For convenience, theinvention will be described hereinafter in terms of the use ofMycobacterium tuberculosis as the first bacterium.

The first bacterium can be present in any suitable form of sample fromthe patient, eg. sputum or other mucal secretions. However, theinvention can be applied to samples of other bodily materials, forexample, blood, urine or stools, from humans, other mammals, such ashorses, cattle, sheep and pigs, and samples from vegetable matter, forexample leaf or root crops or fruit. For convenience, the invention willbe described hereinafter in terms of testing a sample of sputum from ahuman patient.

Typically, the sample will exist as a freshly drawn fluid in which thebacterium is still viable. However, where the sample has been frozen ordried for storage and/or transport prior to testing, it will usually bedesired to hold the sample in a suitable culture medium at about 20 to55° C., preferably 37° C., to ensure that the bacteria are viable andcapable of infection with phage particles in step b of the method of theinvention. If desired, the initial sample can be treated, for examplethinned with N-acetyl cysteine and/or treated with alkali, surfactant oran acid, to reduce the population of other bacteria which mightinterfere with the detection of the bacterium under assessment. Suchpre-treatments can be carried out using conditions and techniquescommonly used in the preparation of bacterial samples for assessment.However, the need to decontaminate the initial sample from otherbacteria is reduced as compared with conventional methods for assessingslow growing bacteria, since the method of the invention is more rapidthan conventional test methods and the effect of the other bacteria isless marked.

The initial sample contains the bacterium to be assessed in a viablestate. This will usually be in a fluid or gel, eg. agar, cultivationmedium. However, the carrier medium could be a particulate solid or thepores of a foraminous material, such as a ceramic frit or a foamedplastic or a filter paper or the mesh apertures in a reticulatematerial, impregnated with a suitable nutrient composition. Forconvenience, the invention will be described hereinafter in terms of theuse of a fluid carrier medium for the initial bacterium sample which issubsequently applied to a gel carrier medium.

The initial sample is infected with a suitable phage which is specificto the type of first bacterium under assessment and to the secondbacterium in which the infected first bacterium is to be cultivated instep c of the method of the invention. The phage may be genericallyspecific to the type of bacterium, so that the phage will infect a rangeof bacteria within a genus. However, it may be preferred to use anarrowly specific phage where a specific bacterium is to be assessed. Ifdesired, the phage may be modified to increase the specificity of thephage, so as to reduce infection of other bacteria in the sample beingassessed. Suitable phage modification can be achieved by knowntechniques, for example by genetic modification, having regard to thespecific bacterium to be infected, for example as described in PCTApplication No WO92/02633. Typical phage for present use include AG1,B1, BG₁, BK₁, C3, D29, D34, D56A, GS4E, HP1, L1, I3 and TM4. The optimumphage for present use will depend upon the first and second bacteriumused, since the phage or its replicand produced upon lysis from abacterial cell needs to be permissive to the bacteria used. Thus, wherethe presence of Mycobacterium tuberculosis is being assessed usingMycobacterium smegmatis as the second bacterium, the phage D29 issuitable; and where Mycobacterium avium is used, the phage TM4 issuitable.

The phage can be provided in the form of, for example, a cultivar in afluid medium or can be provided in the form of freeze or spray driedparticles. The phage is admixed with the viable bacterial sample usingany appropriate technique and under any suitable conditions. Typically,the bacterial sample will be in the form of a fluid and the phageinfection is applied in a fluid carrier to the bacterial sample.

It is preferred to allow the phage infection to proceed at temperaturesof up to 55° C., notably at about 37° C., for from 10 minutes to 4 hoursto allow infection of the majority of the bacterial cells with at leastone phage particle each. However, excessive infection may so weaken thebacteria that they do not survive the in-activation of the non-absorbedphage particles using acid as described below. The optimum infectiontime for a given bacterium can be determined by simple trial and errortests or by microscopic examination of the infected bacteria, havingregard to the conditions to be experienced in the subsequent stages ofthe method of the invention.

The infected bacterial sample is then treated to remove or in-activateany exogenous phage particles which have not been absorbed into thebacterial cells and which remain outside the bacterial cells and exposedto the conditions introduced during the in-activation stage--step b. Forconvenience, the term in-activation will be used herein to denotecollectively any process which renders the non-absorbed phage particlessubstantially incapable of replication in the next step of the method ofthe invention. The term thus includes the physical separation or removalof the phage particles from the sample or the de-activation of theparticles. This in-activation can be carried out using any suitabletechnique, for example those described in PCT Application No WO92/02633.Preferred techniques include washing and filtration of the infectedsample to separate the exogenous phage particles physically from theinfected bacterial cells; by treating the infected sample with an acid,surfactant or chemical to kill or render inactive the exogenous phageparticles; or by preferentially absorbing the exogenous phage particlesfrom the infected sample on a suitable substrate which can then beseparated from the bacterial cells.

For convenience, the invention will be described hereinafter in terms ofchemical or acid treatment of the mixture of infected bacterial cellsand exogenous phage particles. Such treatment is preferably carried outusing a mineral acid or a iron salt thereof, for example ferrousammonium sulphate, or phosphoric or sulphuric acid; a C₁₋₄ aliphaticcarboxylic acid, notably acetic acid, which may be diluted glacialacetic acid or vinegar; or an acidic buffer medium. The treatmentachieves a pH value in the bacterial carrier medium which in-activatesthe phage used, but which does not kill the infected first bacterium.The optimum washing and pH conditions will depend upon the bacteriumunder assessment and can be determined by simple trial and error tests.Typically, the infected bacterial material is acidified and incubated atan elevated temperature, typically about 37° C., for sufficient time,typically about 15 minutes, and the pH is then adjusted to aboutneutrality by the addition of an alkali or base.

In an alternative technique, the exogenous phage is killed by theaddition of a virucide to the infected bacterial material. The optimalvirucide will depend upon the phage to be killed and the nature of thefirst bacterium and can readily be determined by simple trial and errortests. Thus, suitable virucides for present use where the firstbacterium is Mycobacterium tuberculosis include iron salts of mineralacids, and unsaturated fatty acids and alcohols containing from 12 to 24carbon atoms, optionally with actinic or other radiation, notably UVradiation at about 400 to 600 nm wave length.

After in-activation, the bacterial material contains viable firstbacterial cells having inside at least some of them viable phageparticles. This mixture is mixed with a cultivation mixture containing asecond bacterium or mixture of bacteria. These second bacteria arecharacterised in that they are infectable by the phage particles withinthe cells of the first bacteria, that is they are permissive to thephage; and in that they propagate at a rate which is greater than thepropagation rate of the first bacterium under assessment. It ispreferred that the second bacterium has a doubling rate which is atleast twice the doubling rate of the first bacterium. The optimalrelative rate of propagation will depend upon the actual rate ofpropagation of the first bacterium, in that a high relative rate ofpropagation will be required with a very slow growing first bacterium ifthe time taken to produce a detectable phage population and/or reducedsecond bacterial growth areas or plaques is not to be excessive.Typically, the second bacterium will have a doubling rate of from 4 to20 times that of the first bacterium. Thus, for the assessment ofMycobacterium tuberculosis in the initial sample, we have found that theuse of Mycobacterium smegmatis as the second bacterium givesparticularly rapid results. Other suitable second bacteria for presentuse include other Mycobacteria, for example Mycobacterium aurum.

If desired, the infected first bacterium can be allowed to stand afterthe in-activation step b to achieve at least one generation ofreplication and release of the phage particles from the infected cells,before admixture with the second bacterium. Such standing allows thereplicated phage particles to be released from the infected bacterialcells so as to provide more sites of potential infection within thesecond bacterial cells which assists the detection of low numbers ofviable first bacterium in the initial sample.

In step c of the method of the invention, the infected first bacteriumis cultivated in the presence of the second, faster propagatingbacterium. When the phage particles replicate within the infected firstbacterium cells, they form a larger number of phage particles which arereleased from the infected cells as the cells lyse. Each released phageparticle can infect an adjacent bacterial cell, which may be a first orsecond bacterium cell. Each of the phage particles in those newlyinfected bacterial cells can replicate and are released to infectfurther bacteria cells; and so on.

Cultivation of the mixture of first and second bacteria can be carriedout using any appropriate techniques and conditions. Typically, thesecond bacteria will be used in the form of a cultivation in a fluid orgel medium which is mixed with the infected first bacteria. However, thesecond bacteria may be used in the form of a freeze or spray driedpowder or as solid particles having the bacteria impregnated or coatedthereon. For convenience, the invention will be described hereinafter interms of the application of the second bacterium in an agar gel carrierto the infected first bacterium. The amount of the second bacteriumadded to the treated first bacterium sample can vary over wide rangesdepending upon the expected amount of first bacterium cells in thesample and the desired time within which a result from the method of theinvention is required. In general, it is preferred to provide a greaternumber of second bacterium cells, typically from 2 to 50 times, than thenumber of first bacterium cells so as to enhance the probability that aphage particle lysed from an infected first bacterium cell will infect asecond bacterium cell rather than another first bacterium cell. Theoptimum ratio of first to second bacterium cells can readily beestablished by simple trial and error tests.

During the cultivation process, it is preferred that the cultivationmedium change in some detectable way to reflect the presence ofreplicated phage particles, viable second bacteria or plaques of deadbacterial cells due to the action of the phage particles. Typically,growth of the second bacteria will cause cloudiness or colour in an agarcultivation medium and the phage particles and associated dead bacterialcells from which they have been released will result in less cloudy orclear spots, plaques, within the coloured or cloudy medium, so that theplaques can be detected and assessed visually. Alternatively, the areasof differential growth can be detected by differential staining usingconventional staining techniques. Where the cultivation medium containsappropriate ingredients, the bacterial cells can release lumiphores whenthey lyse and these can be detected under UV radiation, for example asdescribed in PCT Application WO92/02633. The increased numbers ofreplicated phage particles can also be detected by immunoassaytechniques, for example ELISA, or another enzymatic technique.

The number of phage particles and/or of plaques gives an indication ofthe number of first bacterium cells present and hence the presence orotherwise of such cells in the initial sample to be assessed.

As indicated above, the method of the invention provides a rapid testfor the presence of an expected bacterial strain in the initial samplefrom the patient. By suitable selection of the phage particles used toinfect the first bacteria, the test can be made specific to a particularbacterium. Alternatively, the test can provide a broad indication as tothe type of bacteria present in the initial sample. The method of theinvention can thus be tailored to the results required. Since theresults can be obtained in a matter of hours rather than days ashitherto required, it is practicable to carry out an initial generalscreen, followed by a specific test once the general type of bacteriumpresent in the initial sample has been identified. Since the method ofthe invention can be carried out using simple cell culture techniquesand the results can be assessed visually, the method of the inventioncan readily be used in third world countries where skilled labour andcomplex laboratory facilities may not be available.

The invention also provides a diagnostic test kit for use in the methodof the invention to detect the presence or absence of a first bacteriumin a sample, which kit comprises a source of a known phage infection forthe first bacterium; a source of a viable second bacterium which has adoubling rate which is faster than that of the first bacterium and whichis permissive to the phage particles to be released from the firstbacterium infected by said phage infection. Preferably also, the kitcomprises means for in-activating and/or removing the exogenous phageparticles from first bacteria infected by said source of phageinfection.

The invention will now be illustrated by the following examples in whichall parts are given by weight unless stated otherwise.

EXAMPLE 1

The rationale of the invention was tested in a safe model system usingslow growing vaccine strain BCG to represent the first bacterium whichhad a typical doubling time of at least 10 hours and fast growingMycobacterium smegmatis as the second bacterium which had a typicaldoubling time of about 2 hours. This example combined liquid and solidagar steps and the exogenous phage were in-activated with acid.

Step 1. A colony of BCG from an agar-based media was grown in liquidculture in 7H9 plus 10% (v/v) OADC supplement (Difco) (referred tohereinafter as 7H9, OADC) at 37° C. for two weeks to yield a stock ofcells which could be used in the detection assay. In the clinicalsituation these cells would originate directly from a clinical sampleand would not require this pre-culture step. In the clinical situationthe slow-growing cells would be Mycobacterium tuberculosis.

Step 2. The liquid culture containing the BCG cells was serially dilutedand 10 μl of each dilution was mixed with 10 μl of 7H9, OADC containing10⁵ D29 bacteriophage (phage). Controls samples were also set upcontaining:

a) no BCG, where 10 μl of media without any BCG was mixed with phage;and

b) a heat-killed control where the BCG was killed by heating at 80° C.for 30 minutes before mixing with the phage.

Step 3. After incubation for 90 minutes at 37° C. exogenous phageparticles were inactivated by addition of 20 μl of 1.2% (v/v) HCl.

Step 4. 20 μl of 22.2 mM NaOH was then added to neutralize the acidity.

Step 5. 10 μl of this solution was then spotted onto an agar platecontaining 7H9, OADC, 5% (v/v) of a stationary phase Mycobacteriumsmegmatis culture and 1.5% Bactoagar (Difco)

Step 6. The plate was then incubated at 37° C. to allow the protectedendogenous phage particles to replicate within the BCG cells. Thereplicated phage particles caused lysis of the BCG cells and werereleased. At least some of the released phage particles infected theMycobacterium smegmatis and set up an infection cycle in these fastergrowing cells.

Step 7. After 16 hours the plates were then observed for clear areas ofphage lysis or plaques among the cloudy growth of uninfectedMycobaterium smegmatis indicator cells. The results were recorded andare shown below:

    ______________________________________                                        Sample          Extent of lysis                                               ______________________________________                                        Undiluted BCG   +                                                               10.sup.-2 dilution 10 plaques                                                 10.sup.-4 dilution -                                                          Heat Killed BCG -                                                             No BCG, phage only  -                                                       ______________________________________                                         + Individual plaques observed but too many to count                           - no plaques                                                             

The heat killed BCG and phage only controls remained negative showingthat live cells were required to provide the phage with protection fromin-activation by acid. Live BCG was able to protect the phage, whichinfected and replicated inside the BCG cells. Upon lysis of the BCGcells, the replicated phage were released and at least some of the phagebegan a replication cycle in the rapid growing Mycobacterium smegmatiswhich yielded plaques after 16 hours of incubation. The highest dilutionof BCG which showed lysis was 10⁻² which, as the original BCG cultureused was still in early log phase, represents approximately 10³ BCGcolony forming units. This assay, therefore, has the potential to detectthe presence of as few as 10³ live slow growing bacteria in a givensample. This sensitivity is higher than that stated for microscopy andabout the same as the sensitivity of culture when the conventionalmethods are applied to the detection of Mycobacteria. However, the timetaken to develop observable plaques was less than 24 hours as comparedto the 4-6 weeks required for conventional culture techniques.

EXAMPLE 2

The procedure of Example 1 was repeated except that the assay wasperformed in a solid agar medium and exogenous phage were removed bywashing rather than in-activated by acid treatment.

Step 1. Step 1 was performed as described in Example 1.

Step 2. Successive 10-fold dilutions of BCG culture were performed in7H9, OADC. 10 μl of each dilution was spotted onto an agar plate (7H9,OADC, 1.5% Bactoagar). 10 μl of a heat killed BCG culture was alsospotted onto a similar plate to provide a control test. The plates wereheld at 37° C.

Step 3. A piece of filter paper was wetted with 7H9, OADC, 3% milkpowder, 10⁶ D29 bacteriophage per milliliter. This was laid on top ofthe spotted plates and incubated at 37° C. for 20 minutes to allowinfection of the bacterial cells on the plates.

Step 4. A stack of five pieces of filter paper was placed on top of thephage soaked filter paper and exogenous phage removed from the environsof the infected cells by capillary action through the stack of filterpaper.

Step 5. When the stack of filter paper was completely wetted all filterpaper was removed from the agar surface and discarded.

Step 6. The infected cells were then stripped from the surface of theagar using a nitrocellulose filter. The nitrocellulose filter was placedon the agar, allowed to wet and then peeled off. This filter was thenplaced cell side down on an agar plate containing the rapid growerMycobacterium smegmatis indicator cells (7H9, OADC, 5% (v/v) of astationary phase Mycobacterium smegmatis culture, 1.5% Bactoagar).

Step 7. The plates from step 6 were incubated at 37° C. until plaquescould be observed visually (about 16 hours) and the results wererecorded.

    ______________________________________                                               Sample    Extent of lysis                                              ______________________________________                                               undiluted BCG                                                                           +++                                                            10.sup.-1 dilution       +++                                                  10.sup.-2 dilution       ++                                                   10.sup.-3 dilution       +                                                    10.sup.-4 dilution       -                                                    Heat Killed BCG               -                                             ______________________________________                                         +++ complete lysis of indicator cells (no cell growth)                        ++ plaques merged with some growth of indicator cells between plaques         + Individual plaques observed but too many to count                           - no plaques                                                             

The heat killed control again remained negative showing that live cellswere required to provide the phage with protection from removal bywashing. Live BCG were able once again to protect the phage whichinfected and replicated inside the BCG cells. Upon lysis of the BCGcells the replicated phage were released and at least some of the phagebegan a replication cycle in the rapid grower Mycobacterium smegmatiswhich yielded plaques after 16 hours of incubation. The highest dilutionof BCG which showed lysis was 10⁻³ which, as the original BCG culturewas in the late log phase of growth, represents approximately 10³ BCGcolony forming units. This assay in this format, therefore, has thepotential to detect the presence of as few as 10³ live slow growingbacteria in a given sample.

EXAMPLE 3

This example demonstrates an alternative method for carrying out thebacterial assay.

Processing of sputum samples for the phage infection step:

The whole sputum sample is placed in a sterile 20 ml screw-capped glassbottle.

1. An equal volume of "Decontaminating Agent" is added to the sample todecontaminate and liquify the sample.

2. The diluted sample is mixed by inverting several times and themixture is left to stand for 10 minutes to allow the sputum to liquify.

3. The sample is then centrifuged at 4000×g for 10 minutes to pellet thebacilli.

4. The resultant supernatant liquor is poured off into neat Hycolindisinfectant and disposed of.

5. The bacilli pellet is re-suspended in 20 mls of sterile distilledwater.

6. The suspended bacilli mixture is centrifuged as in stage 4 to pelletthe bacilli.

7. The bacilli pellet is then re-suspended in 1.0 ml of "Growth Media"and allowed to stand for 24 hours at 35-37° C. to provide the sample forthe assay test.

8. Control samples are prepared by adding 1.0 ml of "Growth Media" to afresh tube to provide a in-activation control sample; and by adding 1drop of "Helper Cells" to 1.0 ml of "Growth Media" in a fresh tube toprovide a positive control for the protection of endogenous phage.

Infection of the bacilli with bacteriophage:

9. Using a sterile pastette, 1 drop (50 μl) of "Bacteriophage" is addedto each test sample, including the in-activation control sample. Mix byagitation and incubate 3.0 hours at 35-37° C. without shaking. Thepositive control for the protection of endogenous phage is not processedat this time.

10. 2.5 hours after adding bacteriophage to the samples, a pellet ofMycobacterium smegmatis is added to the positive control tube.

11. All the test and control samples are incubated for a total of 3hours each. 2 Drops (100l) of "Inactivation Reagent" are added with asterile pastette to each sample, including the control samples. Thesamples are mixed thoroughly and left to stand for 5 minutes.

12. Using a sterile plastic pastette, "Growth Media" is added to the 5ml graduation mark in each sample tube and the contents mixed andincubated for 4 hours at 35-37° C. without shaking.

Visualization of bacteriophage plaques:

13. Using a sterile plastic pastette, 1.0 ml of "Helper Cells" is addedto each test and control sample.

14. 5 ml of molten base agar is added to each sample and the sample andagar mixed by gently swirling.

15. The contents of each tube are poured into a cooled correspondinglylabelled "Indicator Plate".

16. The "Indicator Plates" are left on the bench for 30 minutes to letthe agar solidify.

17. The "Indicator Plates" are held at 37° C. for 15 hours.

18. The number of plaques on each "Indicator Plate" is counted visuallyby observing the plate against a dark background. On the plates thegrowth of the "Helper Cells" is seen as an opaque area with areas oflysis due to bacteriophage being seen as clear or transparent areaswithin this turbidity (plaques).

Checking the control tubes:

Before analysing the results in the test samples, the in-activationcontrol samples are examined for the occurrence of lysis by phage whichhave survived the in-activation treatment. There should be less than 10plaques on this plate. If there are more than 10 plaques, then thechemical inactivation of exogenous phage was incomplete and the assaymust be repeated. The positive control plate is examined for plaques.Extensive lysis should have occurred and there should be large numbersof plaques on this plate. In some circumstances, there may be no growthof "Helper Cells" at all due to complete lysis of the cells with theresult that the plate may have no bacterial lawn.

Each of the test sample plates is examined and the presence of plaquesor lysis is assessed according to the following criteria:

+++ Complete lysis, no bacterial debris

++ Complete lysis but some bacterial debris

+ Plaques merged but some individual plaques if the number of plaquescan be counted record this number

- No plaques observed.

Those plates showing lysis indicate that those sputum samples containviable mycobacteria which have hosted the replication ofmycobacteriophage.

Eight decontaminated sputum samples from pulmonary tuberculosis patientswere tested by the above phage assay technique which showed that five ofthe samples contained viable Mycobacterium tuberculosis and that threedid not. When these results were compared with those obtained byconventional bacterial culturing of the samples, the five samples whichwere positive by culture were also positive by the phage assay, ie.supported survival and replication of the bacteriophage, whereas thethree samples which were negative by culture were also negative by thephage assay, ie. did not support survival and replication of thebacteriophage.

The reagents used in the above test method were as follows:

Decontaminating Agent: 2 gms NaOH, 0.5% N-acetyl L-cysteine in steriledistilled water to 100 ml.

Growth Media: 7H9, OADC supplement with 0.1% glycerol.

Base agar: Growth media with 1.5% Bactoagar added

Bacteriophage: Bacteriophage D29 prepared from a plate lysate. A 90 mmpetri dish showing confluent lysis was flooded with 10 ml 7H9, OADC,0.1% glycerol, 1 mM CaCl₂ and left overnight (16 hours) in the fridge.The liquid was harvested from the plate using a pastet and centrifugedat 4000×g for 15 minutes. The supernatant liquor was filter sterilizedthrough a 0.4 μ filter, aliquotted and stored at 4° C.

Helper Cells: Mycobacterium smegmatis grown to stationary phase andstored at 4° C.

Inactivating Reagent: 100 mM ferrous ammonium sulphate, filtersterilised.

EXAMPLE 4

The rationale of the invention was tested for drug susceptibilitytesting of Mycobacteria in a safe model system. BCG was used as themodel for a susceptible Mycobacteria (susceptible to low concentrationsof isoniazid and rifampicin) whereas Mycobacterium smegmatis, which isinherently resistant to isoniazid and partially resistant to rifampicin,was used as the model for resistant or semi-resistant Mycobacteria.

Step 1. A colony of BCG from an agar-based medium was grown in liquidculture in 7H9, OADC at 37° C. for two weeks to yield a stock of drugsensitive cells which could be used in the detection assay. Similarly astock of Mycobacterium smegmatis was grown for 24 hours in 7H9, OADC toyield a stock of drug resistant cells. In the clinical situation thesecells would originate directly from a clinical sample and would notrequire this pre-culture step. In the clinical situation the drugsensitive cells would be various strains or isolates of Mycobacteriumtuberculosis.

Step 2. 100 μl of each culture was incubated for 72 hours with isoniazidand rifampicin at various concentrations in a volume of 3 mls 7H9, OADC.This step is necessary to allow the cells to be affected by theantibiotic drug. Controls containing no drug were also set up.

Step 3. 100 μl of each culture was then removed for testing.

Step 4. 800 μl of 7H9, OADC containing 5×10⁶ D29 phage was added andincubated at 37° C. either for 4.5 hours for the BCG cultures or for 40minutes for the Mycobacterium smegmatis cultures. Heat killed BCG andMycobacterium smegmatis and phage only samples were also included in theexperiment as controls.

Step 5. Exogenous phage were then in-activated by the addition of 100 μlacid buffer pH 2.2 (made by mixing 32.2 ml of 0.1M disodium citrate and67.8 ml 0.1M HCl).

Step 6. After 5 minutes, 84 μl of 1.0M NaOH was added to neutralize theacidity.

Step 7. A further 4 mls of 7H9, OADC was added and the Mycobacteriumsmegmatis cultures were plated directly. The BCG cultures, however, wereincubated at 37° C. for 3 hours to allow the endogenous, protected,phage to replicate within any live or viable cells.

Step 8. 100 μl of each culture was then mixed with 5 ml of fluid 7H9,OADC containing 5% (v/v) of a stationary phase Mycobacterium smegmatisculture and 0.7% Bactoagar (Difco) and poured into a 50 mm petri dish.

Step 9. After the agar had set, the plates were incubated at 37° C. for16 hours. During this time any endogenous phage surviving the acidtreatment in viable Mycobacteria replicated and lysed the originaltarget cells and at least some of these phage established newreplicative cycles within the rapid grower Mycobacterium smegmatisindicator cells.

Step 10. The plates were then observed for clear areas of phage lysis orplaques among the cloudy growth of uninfected indicator cells. Theresults were recorded and are shown below:

    ______________________________________                                               Isoniazid                                                                              Rifampicin                                                      concentration    Concentration                                                (μg/ml)  (μg/ml)         no    no     Heat-                                  0.1  1.0   10    10  50  100  drug drug Killed                         ______________________________________                                        M. tuberculosis                                                                        122    40    2   4   4   10   71   109  1                              M. smegmatis  ++  ++ + ++ 50  8 ++ ++ 1                                     ______________________________________                                    

This table shows the number of plaques observed with the differentMycobacteria grown in the presence of various antibiotics.

++ plaques merged with some growth of indicator cells between plaques

+ individual plaques observed but too many to count

The slow growing Mycobacterium tuberculosis is susceptible to isoniazidand rifampicin. When grown in the presence of rifampicin, the cells arekilled and consequently there are no viable cells to protect the phagefrom inactivation and to support their growth. The numbers of plaquesobserved in the presence of all concentrations of rifampicin are notsignificantly above background levels; background being the number ofplaques observed in the heat killed samples. The rapid growerMycobacterium smegmatis is, however, resistant to low concentrations ofrifampicin and this can be observed from the large numbers of plaques atthe lowest rifampicin concentration. As the concentration of rifampicinis increased the cells are affected and there is a progressive reductionin the numbers of plaques.

The effect of rifampicin in Mycobacteria is known to be rapid, whereasisoniazid takes some time to exert its effect. Consequently, at thelowest concentration of isoniazid the BCG was unaffected and this wasreflected in the number of observed plaques which was in the region ofthe numbers of plaques observed in the no drug controls. At higherconcentrations of isoniazid, however, there was a marked reduction inthe numbers of plaques reflecting the death of these cells. In contrast,the Mycobacterium smegmatis was mostly unaffected by isoniazid with onlya slight reduction in the numbers of plaques at the highestconcentration.

This experiment shows that this invention can be used to assess the drugsusceptibility of a slow growing cell such as Mycobacteriumtuberculosis. The assay is simple to perform, can be performed on smallnumbers of cells and the result is available within 4 days, whichincludes the time required to allow the drug to exert its effect on thecells. Conventional drug susceptibility assays using culture techniquesneed larger numbers of cells and can take up to 4 weeks to achieve aresult. The Bactec system, which is more rapid than conventional culturetechniques, requires a large item of expensive equipment, whereas themethod of the invention needs the minimum of equipment, is inexpensiveand more suited to the third-world environment where tuberculosis ismore common.

Accordingly, the present invention also provides a method for assessingthe effect of a treatment upon a bacterium, which method comprises:

i. exposing the bacterium to the treatment; and then

ii. assessing the number of viable bacterium cells remaining in a sampleof the bacterium by:

a infecting the sample of the treated bacterium with a phage infectionto which the bacterium is permissive;

b in-activating the exogenous phage particles in the infected sample;

c cultivating the in-activated sample in the presence of a secondbacterium which has a doubling rate greater than that of the firstbacterium, and

d assessing the extent of plaque formation and/or of second bacteriumgrowth.

The method of the invention can also be used to assess the effect of acomposition on a phage by:

a. treating the phage particles with the composition;

b. infecting a first bacterium permissive to the live phage with thetreated phage particles;

c. in-activating the phage particles exogenous to the infected firstbacterium cells;

d. cultivating the in-activated cells in the presence of a secondbacterium which has a doubling rate greater than, preferably at leasttwice as great, typically 4 to 10 times greater, than that of the firstbacterium; and

e. assessing the extent of plaque formation and/or of second bacteriumgrowth in the cultivated second bacterium cells.

In this application of the method of the invention, the extent ofbacterial growth indicates the efficacy or otherwise of the initialtreatment of the phage particles. If no plaques are formed, the initialtreatment has killed or in-activated the phage particles and has thusprevented their infection of the first bacterium cells. The replicationof these cells in the stage of cultivation of the second bacterium hasnot released phage particles which have killed or prevented replicationof the second bacterium cells. The cultivated second cells will thusshow substantially uniform colour or opacity indicating uniform growthof the bacterial cells. Where the composition has not been effective inkilling or in-activating the phage particles, these will infect thefirst bacterium cells and will be protected against the in-activationtreatment of the infected first bacterium. When the infected cells arecultivated in the presence of the second bacterium, the phage particleswill replicate and lyse from the infected first cells to infect thesecond bacterium cells to give plaques or visibly uneven growth of thesecond bacterium. The extent of the plaques or uneven growth gives anindication of the number of phage particles present and hence the extentof the efficacy of the initial treatment on the phage particles. Whilstthis application of the method of the invention may not givequantitative results, it provides a rapid initial screening techniquewhich can be used to discriminate between potential candidatecompositions which can then be subjected to other tests to determine thequantitative effect of the compositions.

Accordingly, in its broadest aspect, the present invention provides amethod for enhancing the time of response of an assay for a firstbacterium, characterised in that:

a. the said first bacterium is exposed to infection by phage particlesto which the said first bacterium is permissive;

b. the infected bacterium is treated to in-activate exogenous phageparticles;

c. the treated bacterium is cultivated in the presence of a secondbacterium which is also permissive to the said phage or its replicandand which has a doubling rate greater than, preferably at least twice asgreat, typically 4 to 10 times greater, than the effective doubling rateof the first bacterium; and

d. assessing the extent of plaque formation and/or of second bacteriumgrowth in the cultivated second bacterium cells.

I claim:
 1. A method for reducing the time of response of an assay for afirst bacterium, wherein:a) the first bacterium is exposed to infectionby phage particles to which said first bacterium is permissive; b) theinfected bacterium is treated to inactivate exogenous phage particles;c) the treated bacterium is cultivated in the presence of a secondbacterium which is permissive to said phage or its replicand and whichhas a doubling rate greater than the effective doubling rate of thefirst bacterium; and d) assessing the extent of plaque formation and/orof second bacterium growth in the cultivated second bacterium cells. 2.A method as claimed in claim 1, wherein said method provides a methodfor detecting the presence of a first bacterium in a sample, whichmethod comprises the steps of:a) infecting a viable sample of the firstbacterium cells with a phage which is specific to that bacterium wherebyat least some of the bacterium cells each absorb one or more of thephage particles; b) inactivating or removing the phage particles whichhave not been absorbed by the bacterium cells; c) cultivating theinfected first bacterium cells in a medium containing at least one othersecond bacterium which has a doubling rate greater than the effectivedoubling rate of said first bacterium, whereby at least some of the saidsecond bacterium cells become infected by phage particles released uponthe lysis of said infected first bacterium cells, each of said infectedbacterium cells acting as a source for further infection of a subsequentgeneration of the infection cycle; and d) monitoring the population ofphage particles and/or the reduced growth of the said second bacteriumin step c) to determine the presence of said first bacterium cells inthe sample under test.
 3. A method as claimed in claim 1, wherein saidmethod provides a method for estimating the effect of a virus growthregulating composition or of a virucide composition on particles of aphage by assessing the ability of the phage particles in replicating ina bacterial cell, which method comprises the steps of treating the phageparticles with the composition, anda) infecting a first bacteriumpermissive to the live phage with the treated phage particles; b)inactivating the phage particles exogenous to the infected firstbacterium cells; c) cultivating the infected first bacterium cells inthe presence of a second bacterium which has a doubling rate greaterthan the effective doubling rate of the first bacterium; d) assessingthe extent of plaque formation and/or of second bacterium growth in thecultivated second bacterium cells; and e) correlating (i) the extent ofplaque formation and/or of second bacterial growth with (ii) the extentof plaque formation and/or second bacterial growth in the absence of thevirus growth regulating composition or virucide, to estimate the effectof the virus growth regulating composition or virucide on the phageparticles.
 4. A method as claimed in claim 1, wherein said methodprovides a method for assessing the effect of a treatment upon abacterium, which method comprises:i) exposing the bacterium to thetreatment; and then ii) assessing the number of viable bacterium cellsremaining in a sample of bacterium by:a) infecting the sample of thetreated bacterium with a phage infection to which the bacterium ispermissive; b) inactivating the exogenous phage particles in theinfected sample; c) cultivating the inactivated sample in the presenceof a second bacterium which has a doubling rate greater than theeffective doubling rate of the first bacterium; d) assessing the extentof plaque formation and/or of second bacterium growth; and e)correlating (i) the extent of plaque formation and/or of secondbacterial growth with (ii) the extent of plaque formation and/or secondbacterial growth in the absence of the treatment of bacterium in stepi), to estimate the effect of the treatment on the bacterium.
 5. Amethod as claimed in any one of claims 1 to 4, wherein the firstbacterium has an effective doubling rate which is greater than 10 hoursat 37° C. in a culture medium comprising 90% v/v of the medium known as7H9 and 10% v/v of the nutrient known as OADC.
 6. A method as claimed inany one of claims 1 to 4 wherein the second bacterium has a doublingrate which is at least twice as great as the effective doubling rate ofthe first bacterium.
 7. A method as claimed in claim 6, wherein thesecond bacterium has a doubling rate which is 4 to 10 times greater thanthe effective doubling rate of the first bacterium.
 8. A method asclaimed in claim 7, wherein the first bacterium is a Mycobacterium sppor a Legionella spp.
 9. A method as claimed in claim 8, wherein thefirst bacterium is Mycobacterium tuberculosis.
 10. A method as claimedin claim 8, wherein the second bacterium is Mycobacterium smegmatis. 11.A method as claimed in any one of claims 1 to 4, wherein the firstbacterium has an effective doubling rate of from 2 to 10 hours.
 12. Amethod as claimed in claim 1, wherein the infection of the firstbacterium with the phage particles is carried out at a temperature up to55° C. and for a period of from 10 minutes to 4 hours.
 13. A method asclaimed in claim 1, wherein the phage is AG1, B1, BG₁, BK₁, C3, D29,D34, D56A, GS4E, HP1, L1, I3 or TM4.
 14. A method as claimed in claim 1,wherein the first bacterium is Mycobacterium tuberculosis, the secondbacterium is Mycobacterium smegmatis and the phage is D29 or TM4.
 15. Amethod as claimed in claim 1, wherein the exogenous phage particles areinactivated in step b) by:i) washing and filtration of the infectedbacterial material to separate the exogenous phage particles from theinfected first bacterium cells; or ii) treatment of the infected firstbacterium material with a virucide, an acid or a surfactant to kill orrender inactive the exogenous phage particles; and/or iii)preferentially absorbing the exogenous phage particles from the firstbacterial material onto a substrate which is separated from thebacterial cells.
 16. A method as claimed in claim 1, wherein the secondbacterium is provided in an initial amount in step c) so as to providefrom 2 to 50 times the number of first bacterium cells in the mixture ofinfected first bacterium and second bacterium to be cultivated.
 17. Amethod as claimed in claim 1, wherein the cultivation step c) of themethod is carried out until a the steady state at which furtherbacterial cell growth and lysis ceases is reached.
 18. A diagnostic kitfor use in the method of claim 1 to detect the presence or absence of afirst bacterium in a sample, wherein the kit comprises:i) a source of aknown phage infection for the first bacterium; and ii) a source of aviable second bacterium which has a doubling rate which is greater thanthat of the first bacterium and which is permissive to the phageparticles to be released from the first bacterium infected by said phageinfection.
 19. A diagnostic kit as claimed in claim 18, wherein the kitfurther comprises means for inactivating and/or removing the exogenousphage particles from first bacteria infected by said source of phageinfection.
 20. A method as claimed in claim 8, wherein the firstbacterium is Mycobacterium avium.
 21. A method as claimed in claim 8,wherein the second bacterium is Mycobacterium aurum.
 22. A method asclaimed in claim 1, wherein the first bacterium is Mycobacterium avium,the second bacterium is Mycobacterium smegmatis and the phage is D29 orTM4.